Methods and compositions for the treatment of cancer

ABSTRACT

The invention relates to a Myc dominant negative mutant, called Omomyc, for use in medicine and for use in the prevention and/or treatment of cancer. The invention also refers to a fusion protein comprising Omomyc and pharmaceutical composition thereof and their use in medicine and, in particular, for treatment of cancer.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 6, 2015, isnamed 151106_0206_88098_Substitute_Sequence_Listing_SC.txt and is 21kilobytes in size.

FIELD OF THE INVENTION

The invention relates to the field of cancer and, more particularly, tomethods and compositions for the treatment of cancer using the Omomycpolypeptide and to compositions comprising Omomyc and one or moreanticancer drugs and their use for the treatment of cancer.

BACKGROUND OF THE INVENTION

The ideal cancer drug should target a non-redundant functioncontinuously necessary for tumor maintenance, but dispensable formaintenance and function of any normal tissues. Hence, the most commonlogic is to target gene products that are specifically mutated incancer, on the basis that these mutant molecules would be the likely“drivers” of the cancer and, perhaps, less critical for normal tissues.For these reasons, much attention has focused on cataloguing recurringlesions in specific cancer types. Unfortunately, there are severalproblems to this approach. First, most solid human cancers pass throughepisodes of genomic instability and exhibit a mutational noise that canobscure the “driver” mutations and their attendant effector pathways.Second, cancers are the end result of a process that involvestransitions through multiple evolutionary bottlenecks. Each bottleneckmay require a specific type of mutation whose function is thereafterdispensable for tumor maintenance and, consequently, not a goodtherapeutic target after that point in the tumor's evolution.

Myc is a basic helix-loop-helix leucine zipper (b-HLH-LZ) proteininvolved in growth control and cancer, which operates in a network withthe structurally related proteins Max, Mad and Mnt. Myc/Max dimersactivate gene transcription and induce cell proliferation or apoptosis.Mad/Max and Mnt/Max complexes act as repressors and cause cell growtharrest and differentiation. All dimers recognize the same DNA consensussite, the CACGTG E-box.

Myc is tightly regulated in normal cells, where its levels are higher inproliferating and lower in non-proliferating. Aberrantly high and/orderegulated Myc activity is causally implicated in most cancers andoften associated with aggressive, poorly differentiated and angiogenictumors. The deregulation of Myc expression is due to overexpressionthrough gene amplifications, loss of transcriptional control, impaireddegradation or increased stabilization. This results in aberrantproliferation, increased survival, changes in metabolism, angiogenesisand inflammation, all of which represent major hallmarks of cancer.Multiple studies substantiated the crucial role of Myc in governingintracellular and extracellular aspects of tumorigenesis suggesting thattargeting its function would be therapeutically valuable.

It is known that down-regulation of myc by a BET bromodomain inhibitorresults in the regression of multiple tumor types (Delmore, J. E., eta., 2011, Cell, 146: 904-917). While this approach displays goodpotential, it presents some limitations such as toxicity and numerousoff targets effects.

Many small molecules disrupting the Myc/Max interaction have displayedlow specificity in cellulo (Prochownik, E. V. and Vogt, P. K., 2010,Genes Cancer 1, 650-659).

A Myc inhibitor, however, has yet to become clinically available and itsdesign presents various caveats: first, Myc is a nuclear transcriptionfactor, which is consequently more difficult to reach than membrane orcytoplasmic molecules; second, Myc does not have an enzymatic “activesite” that could be targeted; third, the Myc family comprises 3different proteins, c-, N and L-Myc, which in certain conditions arefunctionally redundant, so all of them require simultaneous inhibition.Furthermore, there have been concerns that Myc inhibition would induceserious side effects by inhibiting proliferation of normal tissues. Forall these reasons, making a Myc inhibitor drug is challenging.

Omomyc is a dominant-negative MYC mutant comprising the b-HLH-LZ domainof Myc and harboring four amino acid substitutions in the leucine zipperof Myc (Soucek, L. et al., 1998, Oncogene 17, 2463-2472; Soucek, L. etal. (2002), Cancer Res 62: 3507-3510). The amino acid substitutionsE61T, E68I, R74Q, and R75N confer altered dimerization specificity tothe protein, which retains the ability to bind its natural partner Maxand to form homodimers and heterodimers with wild type c-, N- and L-Myc.

Because of these properties, Omomyc is able to prevent Myc-dependentgene transactivation functions both in vitro and in vivo by negating theability of Myc to bind its DNA recognition binding site, the E box(Savino, M. et al., 2011, PLoS One 6, e22284; Soucek, L. et al. (2004),Cell Death Differ 11, 1038 1045). At the same time, Omomyc stronglypotentiates Myc-induced apoptosis in a manner dependent on Mycexpression level and thereby strengthens Myc transrepression activity.Omomyc thus prevents Myc binding to promoter E-boxes and transactivationof target genes while retaining Miz-1-dependent binding to promoters andtransrepression. In the presence of Omomyc, the Myc interactome ischannelled to repression and its activity switches from a pro-oncogenicto a tumor-suppressive one.

TRE-Omomyc;CMVrtTA mice, in which Omomyc expression is controlled by atetracycline-responsive promoter element and the widely expressed rtTAtransactivator is driven by a CMV promoter, exhibit high Omomycexpression in most tissues following administration of doxycycline(Soucek et al., 2008, Nature, 455: 679-683). These mice were crossedwith the well-established LSL-Kras^(G12D) murine model of lungtumorigenesis. Just 3 days of Omomyc expression were sufficient to causedramatic shrinkage of the tumors and one week renders the animalsessentially tumor free. Importantly, although other dividing tissues,such as skin, testis and intestine, exhibited significantly decreasedproliferation rates during the treatment, and displayed a certain degreeof atrophy, the mice exhibited no obvious signs of distress or disease.Moreover, the side effects of Myc inhibition resulting from Omomycexpression are completely reversible and disappear upon discontinuationof the treatment.

To date, despite the fact that the expression of Omomyc has proven to bean efficacious Myc inhibiting strategy in vivo, it has been appliedsolely using a gene therapy approach. Indeed, Omomyc is a peptideconsidered too bulky and unfit for delivery to the desired cellularcompartment (Montagne M. et al., PLoS One. 2012; 7:e32172. doi:10.1371/journal.pone.0032172), Savino M. et al., PLoS One. 2011;6:e22284. doi: 10.1371/journal.pone.0022284) and Genes Dev., 2011, 25:895-7. doi: 10.1101/gad.2053311.)

Moreover, Omomyc is predicted to display poor ability to crossphysiological barriers because of its intrinsic physico-chemicalproperties (e.g. hydrophobicity, as predicted using Kyte & Doolittlehydropathy plot, Kyte J., Doolittle R. F. (1982) J. Mol. Biol.157:105-132). In addition, despite the presence of several arginineresidues within the basic region of Omomyc, the most recent algorithmspredicting spontaneous cell-penetration capacity of peptides do notpredict Omomyc to possess such property (Gautam et al. Journal ofTranslational Medicine 2013, 11:74).

Therefore, providing therapeutic approaches for the treatment of cancerbased on b-HLH-LZ domains capable of transducing across the cellularmembrane of eukaryotic cells and inhibiting Myc-dependent genetransactivation would be advantageous.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention refers to a polypeptide of SEQ ID NO: 1or a functionally equivalent variant thereof for use in medicine as wellas for use in the prevention and/or treatment of cancer.

In another aspect the invention refers to a fusion protein comprising:

-   -   (i) the polypeptide of SEQ ID NO: 1 or a functionally equivalent        variant thereof and    -   (ii) a cell-penetrating peptide sequence and/or a nuclear        localization signal.

In another aspect, the invention refers to a pharmaceutical compositioncomprising the fusion protein according to the invention as well as tothe use of the fusion protein in medicine and, in particular, for thetreatment of cancer.

In another aspect, the invention relates to a composition comprisingtogether or separately:

-   -   (i) a polypeptide of SEQ ID NO: 1, a functionally equivalent        variant thereof or a fusion protein according to the invention        and    -   (ii) an antitumoral agent.

In another aspect, the invention refers to a pharmaceutical compositioncomprising the composition of the invention as well as the use of thecomposition of the invention in medicine and, in particular, for thetreatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Fluorescence imaging of A549 cells incubated for 2 h withOmomyc-FITC at 37° C. shows that Omomyc-FITC is localized in the nucleusand cytoplasm (B) Hoescht staining of the nuclei. (C) Phase contrast.

FIG. 2. Cells incubated with 10 uM of either Omomyc or Max* were countedand stained with AnnexinV and PI. (A) Total cell number of PBS, Omomyc-or Max-treated A549. (B) Percentage of dead cells stained with PI. (C)Percentage of live (non-stained) cells. (D) Percentage of AnnexinVpositive cells.

FIG. 3. (A) Circular dichroism (CD) spectra recorded at 20° C. forc-Myc*, Max* and Omomyc purified proteins (32 μM) showing that Omomychas a folded structure more similar to that of Max* than Myc*. (mdeg,millidegrees) (B) Thermal denaturation studied by circular dichroism at1° C./min for c-Myc*, Max* and Omomyc purified proteins showing thatOmomyc has a folded structure more thermally stable than that of Max*.(° m, millidegrees at the specified wavelength of 222 nm).

FIG. 4. Quantification of fluorescence from confocal microscopy imagesof A549 cells fixed with 4% PFA after 2 hours of incubation at 37° C.with Omomyc or Max* at different peptide concentrations (5, 10 and 25μM) (30-80 cells counted per image). AU, arbitrary units.

FIG. 5. Quantification of fluorescence from confocal microscopy imagesof A549 live cells after 20 minutes of incubation at 37° C. with Omomycor Max* (20 μM). AU, arbitrary units.

FIG. 6. Crystal violet staining of A549 and H1650 lung adenocarcinomacells treated with 25 μM Omomyc or Max* peptide for the indicated times.

FIG. 7. Quantification of the inhibition of proliferation by crystalviolet staining of A549 and H1650 lung adenocarcinoma cells treated with25 μM Omomyc or Max* peptides for the indicated times.

FIG. 8. Dose response of A549 cells to Omomyc and Max* by crystal violetstaining quantification.

FIG. 9. Quantification of proliferation of U87 glioma cells treated withOmomyc or Max* peptides at 25 μM.

FIG. 10. (A) Lung of untreated (left) and treated (right) animals 10minutes after intranasal administration of the fluorescent peptideOmomyc (single dose of 37.5 mg/kg). (B) Brain of untreated (left) andtreated (right) animals 10 minutes after intranasal administration ofthe fluorescent peptide Omomyc (single dose of 37.5 mg/kg).

FIG. 11. Treatment of lung adenocarcinoma with PBS or with Omomyc byintranasal administration. (A) Proliferative rate of tumors. (B)Cellular density.

FIG. 12. Measurement of the % tumor area after treatment of lungadenocarcinoma with PBS or with Omomyc by intranasal administration.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have found that, surprisingly,Omomyc is capable of efficiently transducing across the cellularmembrane and translocate to the nucleus, wherein it exerts itstumor-suppressive effect. Therefore, Omomyc is a bona fide ProteinTransduction Domain (PTD). Hence, for the first time Omomyc can be useditself as an anti-Myc drug without the need of either using vehicles fordelivery of the polypeptide to the cytoplasm of the cell or using genetherapy approaches for delivery the nucleic acid encoding Omomyc to thecell. This allows the use of the Omomyc polypeptide for the treatment ofdiseases associated with deregulated cell proliferation such as cancer.The authors of the present invention have surprisingly found that Omomychas several advantages in comparison to the bHLHZ domain of Max (Max*):

-   -   Omomyc shows greater cell penetrating ability in comparison to        Max* in different cell types and at different concentrations        (Example 6)    -   Omomyc is more thermally stable than Max* and this is a clear        advantage for drug design (Example 5)    -   Omomyc is more efficient than Max* in both preventing the growth        of cells (Example 8) and increasing death of cancer cells        (Example 4).

In addition, Omomyc is capable of crossing the blood-brain barrier(Example 9 and FIG. 10B) and exerting its therapeutic effect in vivo(Example 10).

Therapeutic Methods Using Omomyc

The present invention provides methods for the treatment of cancer basedon the use of a polypeptide having the sequence of SEQ ID NO: 1, whichcorresponds to Omomyc, or of a functionally equivalent variant thereof.

In a first aspect, the invention refers to a polypeptide of SEQ ID NO: 1or a functionally equivalent variant thereof for use in medicine.

In another aspect, the invention refers to a polypeptide of SEQ ID NO: 1or a functionally equivalent variant thereof for use in the preventionand/or treatment of cancer.

In another aspect, the invention also refers to a method for theprevention and/or treatment of cancer that comprises administering to asubject in need thereof a therapeutically effective amount of thepolypeptide of SEQ ID NO: 1 or a functionally equivalent variantthereof.

In another aspect, the invention also refers to the polypeptide of SEQID NO: 1 or a functionally equivalent variant thereof for thepreparation of a medicament for the prevention and/or treatment ofcancer.

The polypeptide of sequence SEQ ID NO: 1 corresponds to the Omomycprotein sequence. The term “Omomyc” as used herein, refers to apolypeptide which consists of a mutated version of the bHLHZip domain ofthe Myc carrying the E61T, E68I, R74Q and R75N mutations (wherein thenumbering of the mutated positions is given with respect to the sequenceof Myc region corresponding to amino acids 365-454 of the polypeptide asdefined under accession number NP_002458 in the NCBI database, releaseof Jun. 27, 2012). The sequence of c-Myc provided in the NCBI databaseunder the accession number NP_002458 is shown below, wherein the regionfrom which Omomyc derives is shown underlined:

(SEQ ID NO: 2)   1mdffrvvenq qppatmplnv sftnrnydld ydsvqpyfyc deeenfyqqq qqselqppap  61sediwkkfel lptpplspsr rsglcspsyv avtpfslrgd ndggggsfst adqlemvtel 121lggdmvnqsf icdpddetfi kniiiqdcmw sgfsaaaklv seklasyqaa rkdsgspnpa 181rghsvcstss lylqdlsaaa secidpsvvf pyplndsssp kscasqdssa fspssdslls 241stesspqgsp eplvlheetp pttssdseee qedeeeidvv svekrqapgk rsesgspsag 301ghskpphspl vlkrchvsth qhnyaappst rkdypaakrv kldsvrvlrq isnnrkctsp 361rssdteenvk rrthnvlerq rrnelkrsff alrdqipele nnekapkvvi lkkatayils 421vqaeeqklis eedllrkrre qlkhkleqlr nsca

The polynucleotide encoding Omomyc (SEQ ID NO: 3) and the correspondingpolypeptide sequence (SEQ ID NO: 1) is shown below, wherein theunderlined and bold triplets correspond to those positions which aremutated with respect to Myc:

¹ACC GAG GAG AAT GTC AAG AGG CGA ACA CAC AAC GTC  TTG GAG CGC CAG ¹Thr Glu Glu Asn Val Lys Arg Arg Thr His Asn Val  Leu Glu 

⁴⁹AGG AGG AAC GAG CTA AAA CGG AGC TTT TTT GCC CTG  CGT GAC CAG ATC

 

 Phe Ala Leu  Arg Asp Gln Ile⁹⁷CCG GAG TTG GAA AAC AAT GAA AAG GCC CCC AAG GTA  GTT ATC CTT AAA ³³Pro Glu Leu Glu Asn Asn Glu Lys Ala Pro Lys Val  Val Ile Leu Lys¹⁴⁵AAA GCC ACA GCA TAC ATC CTG TCC GTC CAA GCA GAG   ACG CAA AAG CTC  ⁴⁹Lys Ala Thr Ala Tyr Ile Leu Ser Val Gln Ala Glu   Thr  Gln Lys Leu¹⁹³ATT TCT GAA ATC GAC TTG TTG CGG AAA CAA AAC GAA   CAG TTG AAA CAC ⁶⁵Ile Ser Glu  Ile  Asp Leu Leu Arg Lys  Gln Asn  Glu  Gln Leu Lys His²⁴¹AAA CTT GAA CAG CTA CGG AAC TCT TGT GCG TAA ⁸¹Lys Leu Glu Gln Leu Arg Asn Ser Cys Ala End

Omomyc also contains the M2 domain of c-Myc, having the sequenceRQRRNELKRSF (SEQ ID NO: 49) (see Dang and Lee, Mol. Cell. Biol., 1988,8:4048-4054) (double underlined above), and which corresponds to anuclear localization signal.

Omomyc is characterized in that it shows increased dimerization capacitywith all three oncogenic Myc proteins (c-Myc, N-Myc and L-Myc). Omomyccan derive from the bHLHZip domain of any Myc protein known in the art,provided that the mutations which result in the tumor suppressor effectare preserved. Thus, the Omomyc that can be used in the presentinvention may derive from any mammal species, including but not beinglimited to domestic and farm animals (cows, horses, pigs, sheep, goats,dog, cats or rodents), primates and humans. Preferably, the Omomycprotein is derived from human Myc protein (accession number NP 002458,release of Jun. 27, 2012).

The term “Myc”, as used, herein, refers to a family of transcriptionfactors which includes c-Myc, N-Myc and L-Myc. Myc protein activatesexpression of many genes through binding on consensus sequence CACGTG(Enhancer Box sequences or E-boxes and recruiting histoneacetyl-transferases or HATs). However, Myc can also act as atranscriptional repressor. By binding the Miz-1 transcription factor anddisplacing p300 co-activator, it inhibits expression of Miz-1 targetgenes. Myc also has a direct role in the control of DNA replication.

The Myc b-HLH-LZ or Myc basic region helix-loop-helix leucine zipperdomain refers to a region which determines Myc dimerization with Maxprotein and binding to Myc-target genes. This region corresponds toamino acids 365-454 of human Myc and is characterized by two alphahelices connected by a loop (Nair, S. K., & Burley, S. K., 2003, Cell,112: 193-205).

The term “functionally equivalent variant”, when referring to Omomyc,refers to any polypeptide which results from the deletion, insertion oraddition of one or more amino acids with respect to the polypeptide ofSEQ ID NO:1 or which results from the chemical modification of thepolypeptide of SEQ ID NO: 1 and which substantially preserves the tumorsuppressor activity of the Omomyc polypeptide. The skilled person willunderstand that the preservation of the tumor suppressor activity ofOmomyc requires that the variant can dimerize with Myc and inhibit itsactivity once found in the nucleus, that it is capable of translocatingacross the cell membrane and that it is capable of translocating acrossthe nuclear envelope.

Suitable functionally equivalent variants of Omomyc include polypeptidesconsisting essentially of the polypeptide of SEQ ID NO:1. In thiscontext, “consisting essentially of” means that the specified moleculewould not contain any additional sequences that would alter the activityof Omomyc.

Suitable functional variants of the targeting peptide are those showinga degree of identity with respect to the peptide of SEQ ID NO:1 of aboutgreater than 25% amino acid sequence identity, such as 25% 40%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degreeof identity between two polypeptides is determined using computeralgorithms and methods that are widely known for the persons skilled inthe art. The identity between two amino acid sequences is preferablydetermined by using the BLASTP algorithm as described previously [BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894,Altschul, S., et al., J. Mol. Biol. 1990; 215: 403-410]. In a preferredembodiment, the sequence identity is determined throughout the wholelength of the polypeptide of SEQ ID NO:1 or throughout the whole lengthof the variant or of both.

The functionally equivalent variants of the Omomyc polypeptide may alsoinclude post-translational modifications, such as glycosylation,acetylation, isoprenylation, myristoylation, proteolytic processing,etc.

Alternatively, suitable functional variants of the targeting peptide arethose wherein one or more positions within the Omomyc polypeptidecontain an amino acid which is a conservative substitution of the aminoacid present in the Omomyc protein mentioned above. “Conservative aminoacid substitutions” result from replacing one amino acid with anotherhaving similar structural and/or chemical properties. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). Selection of such conservative amino acid substitutionsis within the skill of one of ordinary skill in the art and isdescribed, for example by Dordo et al. et al., (J. Mol. Biol, 1999, 217;721-739) and Taylor et al., (J. Theor. Biol., 1986, 119:205-218).

It will be understood that the functionally equivalent variants ofOmomyc contain mutations at positions corresponding to the mutationsE61T, E68I, R74Q and R75N found in Omomyc derived from human c-Myc. Theposition wherein said mutations have to occur in the functionallyequivalent variant can be determined by a multiple sequence alignment ofdifferent Myc sequences and identified by the alignment of thosepositions corresponding to positions 61, 68, 74 and 75 within thesequence of Omomyc derived from human c-Myc.

A multiple sequence alignment is an extension of pairwise alignment toincorporate more than two sequences at a time. Multiple alignmentmethods align all of the sequences in a given query set. A preferredmultiple sequence alignment program (and its algorithm) is ClustalW,Clusal2W or ClustalW XXL (see Thompson et al. (1994) Nucleic Acids Res22:4673-4680). Once the sequences of c-Myc from different organisms andof the variant are compared (aligned) as described herein, the skilledartisan can readily identify the positions within each of the sequencecorresponding to positions and introduce within the Omomyc variantmutations corresponding to the E61T, E68I, R74Q and R75N mutations foundin Omomyc derived from human c-Myc.

Suitable assays for determining whether a polypeptide can be consideredas a functionally equivalent variant of Omomyc include, withoutlimitation:

-   -   Assays which measure the capacity of the polypeptide to form        dimeric complexes with Max and Myc, such as the assays based on        the expression of a reporter gene as described in Soucek et al.        (Oncogene, 1998, 17: 2463-2472) as well as PLA (protein Ligation        assay) or Co-immunoprecipitation.    -   Assays which measure the capacity of the polypeptide to bind to        the Myc/Max recognition site within DNA (the CACGTG site), such        as the electrophoretic mobility shift assay (EMSA) described in        Soucek et al. (supra.)    -   Assays which measure the capacity to repress Myc-induced        transactivation, such as the assay based on the expression of a        reporter gene under the control of the DNA binding sites        specific for Myc/Max as described by Soucek et al. (supra.).    -   Assays based on the capacity of the polypeptide to inhibit        growth of cells expressing the myc oncogene, as described by        Soucek et al. (supra.).    -   Assays which measure the ability of the polypeptide to enhance        myc-induced apoptosis, such as the assays described by Soucek et        al. (Oncogene, 1998: 17, 2463-2472). Moreover, any assay        commonly known in the art for assessing apoptosis in a cell can        be used, such as the Hoechst staining, Propidium Iodide (PI) or        Annexin V staining), trypan blue, DNA laddering/fragmentation        and TUNEL.

In a preferred embodiment, a polypeptide is considered a functionallyequivalent variant of Omomyc if it shows an activity in one or more ofthe above assays which is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 100% of the native Omomyc.

Additionally, functionally equivalent variants of Omomyc are alsocapable of transducing cells after the variant is contacted with saidcell. It will be understood that functionally equivalent variants ofOmomyc contain the protein transducing domain found in native Omomyc oranother functional protein transducing domain.

The term “cell penetrating peptide sequence” is used in the presentspecification interchangeably with “CPP”, “protein transducing domain”or “PTD”. It refers to a peptide chain of variable length that directsthe transport of a protein inside a cell. The delivering process intocell commonly occurs by endocytosis but the peptide can also beinternalized into cell by means of direct membrane translocation. CPPstypically have an amino acid composition that either contains a highrelative abundance of positively charged amino acids such as lysine orarginine or has sequences that contain an alternating pattern ofpolar/charged amino acid and non-polar, hydrophobic amino acids.Examples of CPPs which can be used in the present invention include,without limitation, the CPP found in Drosophila antennapedia protein(RQIKIWFQNRRMKWKK. SEQ ID NO:4), the CPP found in the herpesvirussimplex 1 (HSV-1) VP22 DNA-binding protein(DAATATRGRSAASRPTERPRAPARSASRPRRPVE, SEQ ID NO:5), the CPP of Bac-7(RRIRPRPPRLPRPRPRPLPFPRPG; SEQ ID NO: 6), the CPPs of the HIV-1 TATprotein consisting of amino acids 49-57 (RKKRRQRRR, SEQ ID NO: 7), aminoacids 48-60 (GRKKRRQRRRTPQ, SEQ ID NO: 8), amino acids 47-57(YGRKKRRQRRR; SEQ ID NO: 9); the CPP of 5413-PV peptide(ALWKTLLKKVLKAPKKKRKV; SEQ ID NO: 10), the CPP of penetratin(RQIKWFQNRRMKWKK; SEQ ID NO: 11), the CPP of SynB1 (RGGRLSYSRRRFSTSTGR;SEQ ID NO: 12), the CPP of SynB3 (RRLSYSRRRF; SEQ ID NO:13), the CPP ofPTD-4 (PIRRRKKLRRLK; SEQ ID NO: 14), the CPP of PTD-5 (RRQRRTSKLMKR; SEQID NO: 15), the CPP of the FHV Coat-(35-49) (RRRRNRTRRNRRRVR; SEQ ID NO:16), the CPP of BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO: 17), theCPP of HTLV-II Rex-(4-16) (TRRQRTRRARRNR; SEQ ID NO:18), the CPP ofD-Tat (GRKKRRQRRRPPQ; SEQ ID NO:19), the CPP R9-Tat (GRRRRRRRRRPPQ; SEQID NO: 20), the CPP of MAP (KLALKLALKLALALKLA; SEQ ID NO: 21), the CPPof SBP (MGLGLHLLVLAAALQGAWSQPKKKRKV; SEQ ID NO: 22), the CPP of FBP(GALFLGWLGAAGSTMGAWSQPKKKRKV; SEQ ID NO: 23), the CPP of MPG(ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya; SEQ ID NO: 24), the CPP ofMPG(ENLS) (ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya; SEQ ID NO: 25), the CPPof Pep-1 (ac-KETWWETWWTEWSQPKKKRKV-cya; SEQ ID NO: 26), the CPP of Pep-2(ac-KETWFETWFTEWSQPKKKRKV-cya; SEQ ID NO: 27), a polyarginine sequencehaving the structure RN (wherein N is between 4 and 17), the GRKKRRQRRRsequence (SEQ ID NO: 28), the RRRRRRLR sequence (SEQ ID NO: 29), theRRQRRTS KLMKR sequence (SEQ ID NO: 30); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 31);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 32); RQIKIWFQNRRMKWKK (SEQID NO: 33), the YGRKKRRQRRR sequence (SEQ ID NO: 34); the RKKRRQRRsequence (SEQ ID NO: 35); the YARAAARQARA sequence (SEQ ID NO: 36); theTHRLPRRRRRR sequence (SEQ ID NO: 37); the GGRRARRRRRR sequence (SEQ IDNO: 38).

Suitable assays for determining whether a polypeptide preserves the cellmembrane translocation capacity of Omomyc include, without limitation,assays which measure the capacity of the polypeptide to transduce cellsin culture, such as the assay shown in example 3 of the presentinvention. This assay is based on contacting the polypeptide withculture cells and detecting the presence of the polypeptide in anintracellular location. In a preferred embodiment, the detection of thepolypeptide of the invention is performed by fluorescence microscopy.

In a preferred embodiment, a polypeptide is considered as a functionallyequivalent variant of Omomyc if it is capable of transducing a targetcell at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% asefficiently as native Omomyc.

Additionally, functionally equivalent variants of Omomyc are alsocapable of reaching the nuclei of the transduced cells after the variantis contacted with said cell. It will be understood that functionallyequivalent variants of Omomyc contain the NLS found in native Omomyc oranother functional NLS.

The term “nuclear localization signal”, as used herein, refers to anamino acid sequence of about 4-20 amino acid residues in length, whichserves to direct a protein to the nucleus. Typically, the nuclearlocalization sequence is rich in basic amino acids and exemplarysequences are well known in the art (Gorlich D. (1998) EMBO5.17:2721-7). In some embodiments, the NLS is selected from the groupconsisting of the SV40 large T Antigen NLS (PKKKRKV, SEQ ID NO: 39); theNucleoplasmin NLS (KRPAATKKAGQ AKKKK, SEQ ID NO: 40); the CBP80 NLS(RRRHSDENDGGQPHKRRK, SEQ ID NO: 41); the HIV-I Rev protein NLS(RQARRNRRRWE, SEQ ID NO: 42); the HTLV-I Rex (MPKTRRRPRRSQRKRPPT, SEQ IDNO: 43); the hnRNP A NLS (NQSSNFGPMKGGNFGGRSSGPYGGGGQYFKPRNQGGY, SEQ IDNO: 44); the rpL23a NLS (VHSHKKKKIRTSPTFTTPKTLRLRRQPKYPRKSAPRRNKLDHY,SEQ ID NO: 45). In one embodiment of the invention, the nuclearlocalization signal comprises the motif K (K/R)×(K/R) (SEQ ID NO: 46).

Suitable assays for determining whether a polypeptide is a functionallyequivalent variant of Omomyc in terms of its ability to translocateacross the cellular membrane include double labelling of a cell with areagent specific for the polypeptide and with a dye which specificallylabels the nucleus of the cell (such as DAPI or Hoechst dye). Suchassays are shown in Example 6 of the present invention. In a preferredembodiment, the detection of the polypeptide of the invention isperformed by confocal microscopy or by fluorescence microscopy.

In a preferred embodiment, a polypeptide is considered as a functionallyequivalent variant of Omomyc if it is capable of translocating to thenucleus of the target tumor cells at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% as efficiently as native Omomyc.

Suitable functionally equivalent variants include the polypeptidesOmomyc*TAT and Omomyc*LZArg as defined below:

SEQ  ID Name NO: Sequence Omomyc*TAT 47MTEENVKRRTHNVLERQRRNELKRSFFALRDQIP ELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCAGRKKRRQRRR Omomyc*LZArg 48MTEENVKRRTHNVLERQRRNELKRSFFALRDQIP ELENNEKAPKVVILKKATAYILSVQAETQKLISEIDLLRKQNEQLKHKLEQLRNSCARRRRRRLR

According to the invention, the Omomyc polypeptide or the functionallyequivalent variant thereof are used in a method for the prevention ortreatment of cancer in a subject. It will be understood that thepreventive or therapeutic method according to the invention involves thedirect use of the Omomyc polypeptide or of the functionally equivalentvariant thereof. Thus, the preventive or therapeutic methods accordingto the invention do not involve the administration of the nucleic acidencoding Omomyc or the functionally equivalent variant thereof.

“Prevention” is understood as the administration of a polypeptide of SEQID NO: 1 or a functionally equivalent variant thereof according to thefirst aspect of the invention, or of a medicament containing it in aninitial or early stage of the disease, or to also prevent its onset.

The term “treatment” is used to designate the administration of apolypeptide of SEQ ID NO: 1 or a functionally equivalent variant thereofaccording to the first aspect of the invention, or of a medicamentcontaining it to control the progression of the disease before or afterthe clinical signs have appeared. Control of the progression of thedisease is understood as the beneficial or desired clinical resultswhich include but are not limited to reduction of the symptoms,reduction of the duration of the disease, stabilization of pathologicalconditions (specifically avoiding additional impairment), delaying theprogression of the disease, improving the pathological condition andremission (both partial and complete). The control of the progression ofthe disease also involves a prolongation of survival in comparison tothe expected survival if the treatment was not applied.

The term “cancer” is referred to a disease characterized by uncontrolledcell division (or by an increase of survival or apoptosis resistance),by the ability of said cells to invade other neighbouring tissues(invasion) or by the spread to other areas of the body where the cellsare not normally located (metastasis) through the lymphatic and bloodvessels. Depending on whether or not tumours can spread by invasion andmetastasis, they are classified as being either benign or malignant:benign tumours are tumours that cannot spread by invasion or metastasis,i.e., they only grow locally; whereas malignant tumours are tumours thatare capable of spreading by invasion and metastasis. The methodsaccording to the present invention are useful for the treatment of localand malignant tumours. As used herein, the term cancer includes, but isnot limited to, the following types of cancer: breast cancer; biliarytract cancer; bladder cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; hematologicalneoplasms including acute lymphocytic and myelogenous leukemia; T-cellacute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronicmyelogenous leukemia, multiple myeloma; AIDS-associated leukemias andadult T-cell leukemia/lymphoma; intraepithelial neoplasms includingBowen's disease and Paget's disease; liver cancer; lung cancer;lymphomas including Hodglun's disease and lymphocytic lymphomas;neuroblastomas; oral cancer including squamous cell carcinoma; ovariancancer including those arising from epithelial cells, stromal cells,germ cells and mesenchymal cells; pancreatic cancer; prostate cancer;rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,liposarcoma, fibrosarcoma, and osteosarcoma; slun cancer includingmelanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma,and squamous cell cancer; testicular cancer including germinal tumorssuch as seminoma, non-seminoma (teratomas, choriocarcinomas), stromaltumors, and germ cell tumors; thyroid cancer including thyroidadenocarcinoma and medullar carcinoma; and renal cancer includingadenocarcinoma and Wilms tumor. Other cancers will-be known to one ofordinary skill in the art. In a preferred embodiment, the cancer treatedis lung cancer, preferably lung adenocarcinoma, more preferably aKRas-driven lung adenocarcinoma.

The authors of the present invention have also observed that Omomyc orthe functionally equivalent variant thereof is capable of decreasingcell proliferation irrespective of whether the cancer shows increasedexpression or activity of the Myc protein.

A “subject,” as used herein, includes any animal that has a cancer orexhibits a symptom or cancer, or is at risk for having a cancer orexhibiting a symptom of cancer. Suitable subjects (patients) includelaboratory animals (such as mouse, rat, rabbit, or guinea pig), farmanimals, and domestic animals or pets (such as a cat or dog). Non-humanprimates and, preferably, human patients, are included.

The appropriate dosage of Omomyc or of the functionally equivalentvariant thereof to be used in the methods according to the inventionwill depend on different factors such as the type of cancer to betreated, the severity and course of the disease, whether the compositionis administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the peptide orpolypeptide, and the discretion of the attending physician.

The amount of polypeptide of SEQ ID NO:1, of the functionally equivalentvariant thereof is suitably administered to the patient at one time orover a series of treatments. Depending on the type and severity of thedisease, an appropriate dosage level will generally be about 0.01 to 500mg per kg patient body weight per day which can be administered insingle or multiple doses. Preferably, the dosage level will be about 0.1to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kgper day. A suitable dosage level may be about 0.01 to 250 mg/kg per day,about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day.Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50mg/kg per day. For oral administration, the compositions are preferablyprovided in the form of tablets containing 1.0 to 1000 milligrams of theactive ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0,75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0,800.0, 900.0, and 1000.0 milligrams of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Thecompounds may be administered on a regimen of 1 to 4 times per day,preferably once or twice per day.

The polypeptide of SEQ ID NO:1, or of the functionally equivalentvariant thereof may be administered by any type of suitable route, suchas by oral route, topical route, by inhalation or parenteral route sothat the pharmaceutically acceptable excipients necessary for theformulation of the desired dosage form will be included. The preferredroute of administration of said pharmaceutical compositions is theendovenous route. In another embodiment, the route of administration isthe intranasal route.

In one embodiment, the Omomyc or the functionally equivalent variantthereof is prepared with carriers which will protect said polypeptidefrom a rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated administrationsystems. Biodegradable biocompatible polymers such as ethylenevinylacetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters and polylactic acid can be used. The processes forpreparing said formulations will be clear for persons skilled in theart. The materials can also be commercially obtained in Alza Corporationand Nova Pharmaceuticals, Inc.

Despite the fact that Omomyc and functionally equivalent variantsthereof are capable of translocating across biological membranes, it ispossible to formulate Omomyc or any of its functionally equivalentvariants in nanoparticles. The nanoparticles may contribute to preservethe integrity of the polypeptide in the biological fluids until itreaches the target organ. In addition, nanoparticles can also bemodified so as to include moieties which allow the targeting of thenanoparticule to an organ of interest. In this way, Omomyc or thefunctionally equivalent variant thereof will be delivered in theproximity of the target organ, facilitating access of Omomyc to theinterior of the cells where its biological activity is required.

Thus, in another embodiment, Omomyc or any of its functionallyequivalent variants are provided forming part of a nanoparticle.

As used herein, the term “nanoparticle” refers to any material havingdimensions in the 1-1,000 nm range. In some embodiments, nanoparticleshave dimensions in the 2-200 nm range, preferably in the 2-150 nm range,and even more preferably in the 2-100 nm range. Nanoparticles that canbe used in the present invention include such nanoscale materials as alipid-based nanoparticle, a superparamagnetic nanoparticle, a nanoshell,a semiconductor nanocrystal, a quantum dot, a polymer-basednanoparticle, a silicon-based nanoparticle, a silica-based nanoparticle,a metal-based nanoparticle, a fullerene and a nanotube.

Targeted delivery can be achieved by the addition of ligands withoutcompromising the ability of nanoparticles to deliver their polypeptidepayloads. It is contemplated that this will enable delivery to specificcells, tissues and organs. The targeting specificity of the ligand-baseddelivery systems are based on the distribution of the ligand receptorson different cell types. The targeting ligand may either benon-covalently or covalently associated with a nanoparticle, and can beconjugated to the nanoparticles by a variety of methods as discussedherein.

Examples of proteins or peptides that can be used to targetnanoparticles include transferin, lactoferrin, TGF-β, nerve growthfactor, albumin, HIV Tat peptide, RGD peptide, and insulin, as well asothers.

It will be understood that the formulation of Omomyc or of thefunctionally equivalent variant thereof in a nanoparticle is notintended or is not solely intended for facilitating the access of theOmomyc to the interior of the cell but to protect Omomyc fromdegradation and/or for facilitating targeting of the nanoparticle to theorgan of interest.

Omomyc Conjugates and Fusion Proteins Comprising Omomyc

The present invention also provides conjugates which includes a firstregion comprising the polypeptide of SEQ ID NO:1 or a functionallyequivalent variant thereof and a second region comprising a chemicalmoiety that facilitates cellular uptake of the polypeptide. Examples ofmoieties for enhancing cellular uptake include but are not limited to: ahydrophobic group (e.g., a lipid or fatty acid), a protein transducingdomain, and certain metal chelates.

The presence of additional chemical moieties in the Omomyc moleculeresults in conjugates showing increased capability for beingtranslocated across biological membranes with respect to unmodifiedOmomyc, thereby resulting in increased tumor suppressor activity.

Thus, in another aspect, the invention relates to a conjugatecomprising:

-   (i) the polypeptide of SEQ ID NO: 1 or a functionally equivalent    variant thereof, and-   (ii) a chemical moiety that facilitates cellular uptake of the    polypeptide.

The term “conjugate”, as used herein, refers to two or more compoundswhich are covalently linked together so that the function of eachcompound is retained in the conjugate.

In preferred embodiments, the conjugates according to the inventioncomprise at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10 or morechemical moieties that facilitate cellular uptake of the polypeptide orof the functionally equivalent variant thereof.

In one embodiment, the chemical moiety that facilitates cellular uptakeof the polypeptide is a lipid or a fatty acid.

A fatty acid generally is a molecule comprising a carbon chain with anacidic moiety (e.g., carboxylic acid) at an end of the chain. The carbonchain may of a fatty acid may be of any length, however, it is preferredthat the length of the carbon chain be of at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms, andany range derivable therein. In certain embodiments, the length of thecarbon chain is from 4 to 18 carbon atoms in the chain portion of thefatty acid. In certain embodiments the fatty acid carbon chain maycomprise an odd number of carbon atoms, however, an even number ofcarbon atoms in the chain may be preferred in certain embodiments. Afatty acid comprising only single bonds in its carbon chain is calledsaturated, while a fatty acid comprising at least one double bond in itschain is called unsaturated. The fatty acid may be branched, though inpreferable embodiments of the present invention, it is unbranched.Specific fatty acids include, but are not limited to, linoleic acid,oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid,myristic acid, arachidic acid, palmitoleic acid, arachidonic acid.

In a preferred embodiment, the chemical moiety that facilitates cellularuptake of the polypeptide is a cell penetrating peptide sequence, inwhich case, the conjugate is a fusion protein comprising Omomyc or thefunctionally equivalent variant thereof and the cell penetrating peptidesequence.

The term “fusion protein” relates to proteins generated by genetechnology which consist of two or more functional domains derived fromdifferent proteins. A fusion protein may be obtained by conventionalmeans, e.g., by means of gene expression of the nucleotide sequenceencoding for said fusion protein in a suitable cell. It will beunderstood that the cell penetrating peptide refers to a cellpenetrating peptide which is different from the cell penetrating peptidewhich forms part of the polypeptide of SEQ ID NO: 1 or of the afunctionally equivalent variant thereof.

The terms “polypeptide of SEQ ID NO:1”, “functionally equivalent variantof the polypeptide of SEQ ID NO:1” and “cell penetrating peptide” havebeen described in detail in the context of the medical uses of theinvention and are equally applicable in the context of the fusionprotein.

In a preferred embodiment, said cell-penetrating peptide is not theendogenous Omomyc cell penetrating peptide.

In one embodiment, the cell-penetrating peptide sequence is fused at theN-terminus of the polypeptide of SEQ ID NO: 1 or of the functionallyequivalent variant thereof. In another embodiment, the cell-penetratingpeptide is fused at the C-terminus of the polypeptide of SEQ ID NO: 1 orof the functionally equivalent variant thereof.

In preferred embodiments, the fusion proteins according to the inventioncomprise, in addition to the own cell penetrating peptide found in thepolypeptide of SEQ ID NO:1 or of the functionally equivalent variantthereof, at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10 or moreadditional cell penetrating peptides.

In another preferred embodiment, the conjugates or fusion proteins ofthe invention comprise the polypeptide of SEQ ID NO: 1 or a functionallyequivalent variant thereof and further comprise an N-terminal orC-terminal nuclear localization signal. The term “nuclear localizationsignal (NLS)” has been described in the context of the therapeutic usesof the invention and is equally applicable to the fusion proteins of theinvention. It will be appreciated that the additional NLS refers to anNLS which is different to the endogenous NLS found in Omomyc or in thefunctionally equivalent variant thereof. The additional NLS may be thesame or different to the endogenous NLS found in Omomyc or in thefunctionally equivalent variant thereof.

In one embodiment, the NLS is one of the NLS which appears endogenouslyin the Myc sequence, such as the M1 peptide (PAAKRVKLD, SEQ ID NO: 50)or the M2 peptide (RQRRNELKRSF, SEQ ID NO: 49) (see Dang and Lee,supra.).

In preferred embodiments, the conjugates or fusion proteins according tothe invention comprise, in addition to the endogenous NLS found in thepolypeptide of SEQ ID NO:1 or in the functionally equivalent variantthereof, at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10 or NLS.

The skilled person will understand that it may be desirable that thefusion protein further comprises one or more flexible peptides thatconnect the polypeptide of SEQ ID NO:1 or the functionally equivalentvariant thereof, the cell penetrating peptide sequence and/or the NLS.Thus, in a particular embodiment the polypeptide of the invention isdirectly connected to the cell penetrating peptide sequence. In anotherparticular embodiment, the polypeptide of the invention is connected tothe cell penetrating peptide sequence through a flexible peptide. In aparticular embodiment the polypeptide of the invention is directlyconnected to the cell penetrating peptide sequence and to the NLS. Inanother particular embodiment, the polypeptide of the invention isconnected to the cell penetrating peptide sequence through a firstflexible peptide linker and to the NLS through a second flexible peptidelinker.

As used herein, the term “flexible peptide”, “spacer peptide” or “linkerpeptide” refers to a peptide that covalently binds two proteins ormoieties but which is not part of either polypeptide, allowing movementof one with respect to the other, without causing a substantialdetrimental effect on the function of either the protein or the moiety.Thus, the flexible linker does not affect the tumour suppressor activityof the Omomyc sequence, the cell penetrating activity of the cellpenetrating peptide or the nuclear localization capacity of the NLS.

The flexible peptide comprises at least one amino acid, at least twoamino acids, at least three amino acids, at least four amino acids, atleast five amino acids, at least six amino acids, at least seven aminoacids, at least eight amino acids, at least nine amino acids, the least10 amino acids, at least 12 amino acids, at least 14 amino acids, atleast 16 amino acids, at least 18 amino acids, at least 20 amino acids,at least 25 amino acids, at least 30 amino acids, at least 35 aminoacids, at least 40 amino acids, the least 45 amino acids, at least 50amino acids, at least 60 amino acids, at least 70 amino acids, at least80 amino acids, at least 90 amino acids, or about 100 amino acids. Insome embodiments the flexible peptide will permit the movement of oneprotein with respect to the other in order to increase solubility of theprotein and/or to improve its activity. Suitable linker regions includea poly-glycine region, the GPRRRR sequence (SEQ ID NO: 51) ofcombinations of glycine, proline and alanine residues.

In some embodiments the fusion protein of the invention can comprise anadditional chemical moiety including, among others, fluorescence groups,biotin, polyethylene glycol (PEG), amino acid analogs, unnatural aminoacids, phosphate groups, glycosyl groups, radioisotope labels, andpharmaceutical molecules. In other embodiments, the heterologouspolypeptide can comprise one or more chemically reactive groupsincluding, among others, ketone, aldehyde, Cys residues and Lysresidues.

In a particular embodiment, the conjugates or fusion proteins of theinvention comprise a tag bound to the conjugate or to the C-terminal orN-terminal domain of said fusion protein or variant thereof. Said tag isgenerally a peptide or amino acid sequence which can be used in theisolation or purification of said fusion protein. Thus, said tag iscapable of binding to one or more ligands, for example, one or moreligands of an affinity matrix such as a chromatography support or beadwith high affinity. An example of said tag is a histidine tag (His-tagor HT), such as a tag comprising 6 residues of histidine (His6 or H6),which can bind to a column of nickel (Ni2+) or cobalt (Co2+) with highaffinity. His-tag has the desirable feature that it can bind its ligandsunder conditions that are denaturing to most proteins and disruptive tomost protein-protein interactions. Thus, it can be used to remove thebait protein tagged with H6 following the disruption of protein-proteininteractions with which the bait has participated.

Additional illustrative, non-limitative, examples of tags useful forisolating or purifying a fusion protein include Arg-tag, FLAG-tag(DYKDDDDK; SEQ ID NO:52), Strep-tag (WSHPQFEK, SEQ ID NO:53), an epitopecapable of being recognized by an antibody, such as c-myc-tag(recognized by an anti-c-myc antibody), HA tag (YPYDVPDYA, SEQ IDNO:54), V5 tag (GKPIPNPLLGLDST, SEQ ID NO:55), SBP-tag, S-tag,calmodulin binding peptide, cellulose binding domain, chitin bindingdomain, glutathione S-transferase-tag, maltose binding protein, NusA,TrxA, DsbA, Avi-tag, etc. (Terpe K., Appl. Microbiol. Biotechnol. 2003,60:523-525), an amino acid sequence such as AHGHRP (SEQ ID NO:56) orPIHDHDHPHLVIHSGMTCXXC (SEQ ID NO:57), (3-galactosidase and the like.

The tag can be used, if desired, for the isolation or purification ofsaid fusion protein.

In another aspect, the invention relates to the conjugate or fusionprotein according to the invention for use in medicine.

In another aspect, the invention relates to the conjugate or fusionprotein according to the invention for use in the prevention and/ortreatment of cancer.

In another aspect, the invention relates to the use of the conjugate orthe fusion protein according to the invention for the preparation of amedicament for the prevention and/or treatment of cancer.

In another aspect, the invention relates to a method of treatment and/orprevention of cancer in a subject which comprises the administration tosaid subject of the conjugate or of the fusion protein according to theinvention.

In a preferred embodiment the cancer to be prevented or treated isMyc-induced cancer. In another preferred embodiment, the cancer to beprevented or treated is a cancer associated with a mutation in the KRASgene. In one embodiment, the mutation in the KRAS gene is a mutation atthe glycine at position 12, at the glycine at position 13 or at theglutamine at position 61. In a more preferred embodiment, the mutationis selected from the group consisting of the G12S mutation, the G12Vmutation, the G13D mutation, the G12C mutation, the G12R mutation, theG12F mutation, the G12I mutation, the G13C mutation, the G13R mutation,or the Q61L mutation.

The terms “medicament”, “prevention”, “treatment”, “cancer” and“Myc-induced cancer” have been defined previously and equally apply tothis aspect of the invention.

Antitumor Compositions

The unexpected finding that the Omomyc polypeptide is capable oftranslocating across biological membranes and exerting its tumorsuppressor activity when provided as a polypeptide opens the possibilityof formulating Omomyc with other antitumor drugs.

Thus, in another aspect, the present invention also providescompositions containing, together or separately, a first componentselected from the group consisting of:

(i) Omomyc,

(ii) a functionally equivalent variant thereof,

(iii) a conjugate or fusion protein according to the invention

and, as a second component, an antitumor compound.

The first component of the compositions according to the inventionincludes a polypeptide according to SEQ ID NO:1, a functionallyequivalent variant thereof or a fusion protein according to theinvention. Suitable polypeptides, functionally equivalent variants ofthe polypeptide of SEQ ID NO:1 and fusion proteins have been describedabove in the context of the therapeutic methods according to theinvention and are equally applicable to the compositions according tothe invention.

As used herein, “antitumoral agent” is understood as said biological orchemical compound which treat tumors or prevent the formation thereof.In a preferred embodiment said antitumoral agent is selected from thegroup consisting of a cytotoxic agent, an antiangiogenic agent, anantimetastatic agent and an antiproliferative agent.

As used in the present invention, the term “cytotoxic agent” relates toan agent which is capable of promoting cell death and which has capacityfor reducing the growth, stopping the growth or destroying cells and,particularly, rapidly proliferating cells and, yet more particularly,tumor cells. Cell death can be caused by any mechanism, such as forexample apoptosis, although it is not limited to this cause, by themetabolism inhibition, the interference with the organization of thecytoskeleton or the chemical modification of the DNA. The term cytotoxicagent comprises any chemotherapy agent including small organicmolecules, peptides, oligonucleotides and the like; toxins; enzymes;cytokines; radioisotopes or radiotherapy agents.

“Chemotherapy agents” are understood as chemical compounds such as,without limitation, anthracycline antibiotics such as doxorubicin anddaunorubicin, taxanes such as Taxol™ and docetaxel, vinca alkaloids suchas vincristine and vinblastine, 5-fluorouracil (5-FU), leucovorin,irinotecan, idarubicin, mitomycin C, oxaliplatin, raltitrexed,tamoxifen, cisplatin, carboplatin, methotrexate, actinomycin D,mitoxantrone, blenoxane or mithramycin.

“Toxin” is understood as a toxic agent which conjugates with thepolypeptide according to the first aspect of the invention or the fusionprotein according to the second aspect of the invention forming animmunotoxin. The conjugation of determined toxins with said polypeptideor with said fusion protein reduces the toxicity of the former, enablingtheir use as therapeutic agents, because otherwise they would be tootoxic. The binding between the toxin and the polypeptide according tothe first aspect of the invention or the fusion protein according to thesecond aspect of the invention is performed chemically, conserving itsbiological activity. Their separation generally occurs in the lysosomesof the target cells such that the mentioned chemical binding is onlybroken in the enclosed acidic cellular environment provided by thelysosomes. Toxins useful in the context of the present invention areplant toxins, bacterial toxins, toxins of fungal or animal origin andfragments thereof, such as, without limitation, the ricin A-chain,saponin, the diphtheria A-chain, active non-binding fragments of thediphtheria toxin, Pseudomonas aeruginosa exotoxin A-chain, abrinA-chain, modecin A-chain, α-sarcin, Leurites fordii A-proteins, dianthinproteins, Phytolaca americana (PAPI, PAPII and PAP-S) proteins,Momordica charantia inhibitor, curcine, crotin, Saponaria officinalisinhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin andtrichothecenes.

“Enzymes” are understood in the context of the present invention astoxin or drug activating enzymes, such as, without limitation, alkalinephosphatase which activates etoposide and doxorubicin; carboxypeptidaseG2 which activates nitrogen mustards; beta-lactamase which activatesdoxorubicin, paclitaxel and mitomycin.

“Cytokines” are understood as peptides of different sizes and molecularweights which synthesize the cells of the immune system for the purposeof regulating the immune response, and they can be hormones, growthfactors, necrosis factors, etc. They can be of natural origin or fromrecombinant cell cultures and biologically active equivalents of naturalsequence cytokines. Their conjugation with antibodies gives rise toimmunocytokines. Cytokines useful in the present invention are, withoutlimitation, TNF factor alpha, INF-gamma, GM-GSF factor or IL-2.

“Radioisotopes” is understood as radioactive isotopes such as, withoutlimitation, ¹³¹I, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁸Re, ⁶⁷Cu, ²¹¹At, ²¹³Bi, ¹²⁵I, ¹¹¹In.

Antiangiogenic agent” is understood as a chemical or biologicalsubstance which inhibits or reduces the formation of new blood vessels,i.e., angiogenesis.

Antiangiogenic agents that can be conjugated with the polypeptideaccording to the first aspect of the invention or with the fusionprotein according to the second aspect of the invention include, withoutlimitation, an antiangiogenic agent selected from the group ofpaclitaxel, 2-methoxyestradiol, prinomastat, batimastat, BAY 12-9566,carboxyamidotriazole. CC-1088, dextromethorphan acetic acid,dimethylxanthenone acetic acid, endostatin, IM-862, marimastat,penicillamine, PTK787/ZK 222584, RPI.4610, squalamine lactate, SU5416,thalidomide, combretastatin, tamoxifen, COL-3, neovastat, BMS-275291.SU6668, anti-VEGF antibodies, Medi-522 (Vitaxin II), CAI. interleukin12, IM862, amiloride, angiostatin, K1-3 angiostatin, K1-5 angiostatin,Captopril, DL-alpha-difluoromethylomithine,DL-alpha-difluoromethylomithine HCl, endostatin, fumagillin, herbimycinA. 4-Hydroxyphenylretinamide, juglone, laminin, laminin hexapeptide,laminin pentapeptide, lavendustin A, medroxyprogesterone, minocycline,placenta ribonuclease inhibitor, suramin, thrombospondin, antibodiesdirected against proangiogenic factors (for example, Avastin, Erbitux,Vectibix, Herceptin); low molecular weight tyrosine kinase inhibitors ofproangiogenic growth factors (for example Tarceva, Nexavar, Sutent,Iressa); mTOR inhibitors (for example Torisel); interferon alpha, betaand gamma, IL-12, matrix metalloproteinase inhibitors (for example,COL3, marimastat, batimastat); ZD6474, SU11248, vitaxin; PDGFRinhibitors (for example Gleevec); NM3 and 2-ME2; cyclopeptides such ascilengitide.

“Antimetastatic agent” is understood as a chemical or biologicalsubstance which inhibits or reduces metastasis, i.e., the distancepropagation, fundamentally by the lymphatic or blood stream, of thecancer causing cells, and the growth of new tumors in the destinationsites of said metastasis.

“Antiproliferative agent” is understood as a chemical or biologicalsubstance which is capable of preventing or inhibiting the formation orgrowth of tumors. Antiproliferative agents include but are not limitedto (i) antimetabolites such as folic acid antimetabolites (aminopterin,denopterin, methotrexate, edatrexate, trimetrexate, nolatrexed,lometrexol, pemetrexed, raltitrexed, piritrexim, pteropterin,leucovorin, 10-propargyl-5,8-dideazafolate (PDDF, CB3717)), purineanalogs (cladribine, clofarabine, fludarabine, mercaptopurine,pentostatin, thioguanine) and pyrimidine analogs (capecitabine,cytarabine or ara-C, decitabine, fluorouracil, 5-fluorouracil,doxifluridine, floxuridine and gemcitabine) (ii) natural products, suchas antitumor antibiotics and mitotic inhibitors such vinca alkaloidssuch as vindesine, vincristine, vinblastine, vinorelbine; taxanes suchas paclitaxel (Taxol™), docetaxel (Taxotere™); colchicine (NSC 757),thiocolchicine (NSC 361792), colchicine derivatives (e. g., NSC 33410),and allocolchicine (NSC 406042); halichondrin B (NSC 609395); dolastatin10 (NSC 376128); maytansine (NSC 153858); rhizoxin (NSC 332598);epothilone A, epothilone B; discodermolide; estramustine; nocodazole;(iii) hormones and antagonist thereof, such tamoxifen, toremifene,anastrozole, arzoxifene, lasofoxifene, raloxifene, nafoxidine,fulvestrant, aminoglutethimide, testolactone, atamestane, exemestane,fadrozole, formestane, letrozole, goserelin, leuprorelin or leuprolide,buserelin, histrelin, megestrol and fluoxymesterone; (iv) biologicalagents, such as viral vectors, interferon alpha and interleukines; (v)platinum based compounds such as carboplatin, cisplatin[cis-diamminedichloroplatinum, (CDDP)], oxaliplatin, iproplatin,nedaplatin, triplatin tetranitrate, tetraplatin, satraplatin (JM216),JM118 [cis ammine dichloro (II)], JM149 [cis ammine dichloro(cyclohexylamine) trans dihydroxo platinum (IV)], JM335 [trans amminedichloro dihydroxo platinum (IV)], transplatin, ZD0473, cis, trans,cis-Pt(NH3)(C6H11NH2)(OOCC3H7)2Cl,malanate-1,2-diaminociclohexanoplatin(II),5-sulphosalycilate-trans-(1,2-diaminociclohexane)platin (II) (SSP),poly-[(trans-1,2-diaminocyclohexane)platin]-carboxyamilose (POLY-PLAT)and 4-hydroxy-sulphonylphenylacetate (trans-1,2-diaminocyclohexane)platinum (II) (SAP) and the like and (vi) DNA-alkylating drugs such asnitrogen mustards, nitrosoureas, ethylenimine derivatives, alkylsulfonates and triazenes, including, but not limited to,cyclophosphamide (Cytoxan™), busulfan, improsulfan, piposulfan,pipobroman, melphalan (L-sarcolysin), chlorambucil, mechlorethamine ormustine, uramustine or uracil mustard, novembichin, phenesterine,trofosfamide, ifosfamide, carmustine (BCNU), lomustine (CCNU),chlorozotocin, fotemustine, nimustine, ranimnustine, semustine(methyl-CCNU), streptozocin, thiotepa, triethylenemelamine,triethylenethiophosphoramine, procarbazine, altretamine, dacarbazine,mitozolomide and temozolomide.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising theconjugates and fusion proteins of the invention or the compositionsaccording to the invention. Thus, in another aspect, the inventionprovides a pharmaceutical composition comprising a pharmaceuticallyactive amount of the conjugate or fusion protein according to theinvention and a pharmaceutically active carrier (first pharmaceuticalcomposition of the invention). In another aspect, the invention providesa pharmaceutical composition comprising a pharmaceutically active amountof the composition according to the invention and a pharmaceuticallyactive carrier (second pharmaceutical composition according to theinvention).

As it is used in the present invention, the expression “pharmaceuticalcomposition” relates to a formulation that has been adapted foradministering a predetermined dose of one or several therapeutic usefulagents to a cell, a group of cells, an organ, a tissue or an animal inwhich cell division is uncontrolled, such cancer.

The first pharmaceutical composition of the invention contains apharmaceutical effective amount of a conjugate or fusion proteinaccording to the invention. Suitable conjugates and fusion proteins foruse in the pharmaceutical compositions according to the presentinvention include any of the fusion proteins mentioned above under theparagraph Omomyc conjugates and fusion proteins of the invention.

The second pharmaceutical composition of the invention contains apharmaceutical effective amount of a composition according to theinvention and a pharmaceutically active carrier. The secondpharmaceutical composition of the invention comprise the polypeptide ofSEQ ID NO:1, a functionally equivalent variant thereof or a fusionprotein according to the invention. Suitable functionally equivalentvariants of the polypeptide of SEQ ID NO:1 or suitable fusion proteinsfor use in the second pharmaceutical compositions according to theinvention are as defined above under therapeutic uses of the inventionor under fusion proteins of the invention, respectively.

The expression “Pharmaceutical effective amount”, as used herein, isunderstood as an amount capable of providing a therapeutic effect, andwhich can be determined by the person skilled in the art by commonlyused means. The amount of the Omomyc polypeptide, of the functionallyequivalent variant thereof or of the fusion protein or of theantitumoral compound that may be combined in the pharmaceuticalcompositions according to the invention will vary depending upon thesubject and the particular mode of administration. Those skilled in theart will appreciate that dosages may also be determined with guidancefrom Goodman and Goldman's The Pharmacological Basis of Therapeutics,Ninth Edition (1996), Appendix II, pp. 1707-1711 and from Goodman andGoldman's The Pharmacological Basis of Therapeutics, Tenth Edition(2001), Appendix II, pp. 475-493.

The appropriate dosage of the active principle or principles within thepharmaceutical composition will depend on the type of cancer to betreated, the severity and course of the disease, whether the compositionis administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the peptide orpolypeptide, and the discretion of the attending physician. The amountof polypeptide of SEQ ID NO:1, of the functionally equivalent variantthereof, of the fusion protein is suitably administered to the patientat one time or over a series of treatments. Depending on the type andseverity of the disease, an appropriate dosage level will generally beabout 0.01 to 500 mg per kg patient body weight per day which can beadministered in single or multiple doses. Preferably, the dosage levelwill be about 0.1 to about 250 mg/kg per day; more preferably about 0.5to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5or 5 to 50 mg/kg per day. For oral administration, the compositions arepreferably provided in the form of tablets containing 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0,20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0,600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient for the symptomatic adjustment of the dosage to the patientto be treated. The compounds may be administered on a regimen of 1 to 4times per day, preferably once or twice per day.

In the case of the second pharmaceutical compositions according to theinvention, which contain a first component selected from the polypeptideof SEQ ID NO:1, a functionally equivalent variant thereof and a fusionprotein according to the invention and a second component which is anantitumoral agent, the composition may be presented as a singleformulation (for example, as a tablet or a capsule comprising a fixedquantity of each one of the components) or can, on the other hand, bepresented as separate formulations to be later combined for joint,sequential, or separate administration. The compositions of theinvention also include the formulation as a kit-of-parts wherein thecomponents are formulated separately but are packaged in the samecontainer. Those skilled in the art will appreciate that the formulationof the different components in the case of the second pharmaceuticalcomposition according to the invention may be similar, in other words,similarly formulated (in tablets or pills), which allows theiradministration by the same route. In the case where the differentcomponents of the invention are formulated separately, the twocomponents can be presented in a blister. Each blister contains thedrugs that must be consumed during the day. If the drugs must beadministered several times a day, the drugs corresponding to eachadministration can be placed in different sections of the blister,preferably recording in each section of the blister the time of day whenthey should be administered. Alternatively, the components of thecomposition of the invention can be formulated differently so that thedifferent components are differently administered. Thus, it is possiblethat the first component is formulated as a tablet or capsule for itsoral administration and the second component is formulated for itsintravenous administration or vice versa. The ratio between thecomponents that are part of the compositions used in the secondpharmaceutical composition according to the invention can be adjusted bythe skilled person depending on the antitumor agent used in eachparticular case, as well as of the desired indication. Thus, theinvention envisages compositions wherein the ratio between thequantities of the two components can range from 50:1 to 1:50, inparticular from 20:1 to 1:20, from 1:10 to 10:1, or from 5:1 to 1:5.

The first and the second pharmaceutical compositions of the inventioncan also contain one or several additional compounds for the preventionand/or treatment of pathologies in which there is an uncontrolled celldivision, such as cancer. Said additional compounds, such as antitumoralagents can form part of the pharmaceutical composition as independententities.

The first and the second pharmaceutical compositions of the inventionalso contain one or several additional pharmaceutically acceptableexcipients. “Pharmaceutically acceptable excipient” is understood atherapeutically inactive substance said to be used for incorporating theactive ingredient and which is acceptable for the patient from apharmacological/toxicological point of view and for the pharmaceuticalchemist who manufactures it from a physical/chemical point of view withrespect to the composition, formulation, stability, acceptation of thepatient and bioavailability.

The number and the nature of the pharmaceutically acceptable excipientsdepend on the desired dosage form. The pharmaceutically acceptableexcipients are known by the person skilled in the art (Fauli y Trillo C.(1993) “Tratado de Farmacia Galénica”, Luzán 5, S. A. Ediciones,Madrid). Said compositions can be prepared by means of the conventionalmethods known in the state of the art (“Remington: The Science andPractice of Pharmacy”, 20^(th) edition (2003) Genaro A. R., ed.,Lippincott Williams & Wilkins, Philadelphia, US).

The first and the second pharmaceutical compositions of the invention orcan be administered by any type of suitable route, such as by oralroute, topical route, by inhalation or parenteral route so that thepharmaceutically acceptable excipients necessary for the formulation ofthe desired dosage form will be included. The preferred route ofadministration of said pharmaceutical compositions is the endovenousroute.

“Oral route” is understood as the pharmaceutical compositionincorporated into the organism after deglutition. In a particularembodiment, the pharmaceutical composition of the invention can be in adosage form suitable for its administration by oral route, whether it issolid or liquid. The dosage forms suitable for their administration byoral route can be tablets, capsules, syrups or solutions, and cancontain any conventional excipient known in the art, such as binders,for example syrup, acacia, gelatin, sorbitol or polyvinylpyrrolidone;filling agents, for example lactose, sugar, corn starch, calciumphosphate, sorbitol or glycine; lubricants for compression, for example,magnesium stearate; disintegrating agents, for example starch,polyvinylpyrrolidone, sodium glycolate of starch or microcrystallinecellulose; or pharmaceutically acceptable wetting agents such as sodiumlauryl sulfate. The solid oral compositions can be prepared by means ofconventional processes of mixing, filling or compressing. Repetitivemixing operations can be used to completely distribute the active agentin those compositions that use high amounts of filling agents. Saidoperations are conventional in the art. The tablets can be prepared, forexample, by means of wet or dry granulation, and optionally coating themaccording to the processes known in the common pharmaceutical practice,particularly with an enteric coating.

On the other hand, “topical route” is understood as an administration bynon-systemic route, and includes the application of a pharmaceuticalcomposition of the invention externally on the epidermis, in the oralcavity and the instillation of said composition into ears, eyes andnose, and in which it does not significantly enter the blood stream.“Systemic route” is understood as the administration by oral route,intravenous route, intraperitoneal route and intramuscular route. Theamount of antibody required for the therapeutic or prophylactic effectwill naturally vary according to the elected antibody, the nature andthe severity of the illness that is going to be treated, and thepatient.

“Inhalation” is understood as the administration by intranasal route andby oral inhalation. The dosage forms suitable for said administration,such as a formulation in aerosol or a meter dosed inhaler can beprepared by means of conventional techniques. In an embodiment the routeof administration is the intranasal route.

As it is used herein, the term “parenteral”, includes administration byintravenous route, intraperitoneal route, intramuscular route orsubcutaneous route. Subcutaneous, intramuscular and intravenous dosageforms of parenteral administration are generally preferred.

In one embodiment, the first and the second pharmaceutical compositionsof the invention can be adapted for their parenteral administration,such as sterile solutions, suspensions or lyophilized products in theappropriate dosage unit form. The pharmaceutical compositions suitablefor its injectable use include sterile aqueous solutions (when they aresoluble in water), or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. For its administration by intravenous route, some suitablecarriers include saline solution buffered with phosphate (PBS). In allthe cases, the composition must be sterile, and must be fluid to thepoint which that there exists easy ability for being injected. It mustbe stable in the preparation and storage conditions, and must beprotected from the contamination action of microorganisms such asbacteria and fungi. The carrier can be a solvent or a dispersion mediumwhich contains, for example, water, ethanol, a pharmaceuticallyacceptable polyol such as glycerol, propylene glycol, liquidpolyethylene glycol and suitable mixtures thereof. Suitable fluidity canbe maintained, for example, by means of using a coating such aslecithin, by means of maintaining the particle size required in the caseof dispersion and by means of using surfactants. The prevention of theaction of the microorganisms can be achieved by means of variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thiomersal, and the like. In mostcases, it will be preferable to include isotonic agents, for example,sugars; polyalcohols such as mannitol, sorbitol; or sodium chloride inthe composition. The prolonged absorption of the injectable compositionsmay be caused by the inclusion of an agent which delays the absorption,for example, aluminum and gelatin monostearate.

The injectable sterile solutions can be prepared by incorporating theactive compound in the required amount in a suitable solvent with one ora combination of the aforementioned ingredients, as needed, followed bysterilization by filtration through sterile membranes. Generally, thedispersions are prepared by incorporating the active compound in asterile vehicle containing a basic dispersion medium and the rest of theingredients required from among those previously listed. In the case ofsterile powders for the preparation of injectable sterile solutions, thepreferred preparation processes are vacuum drying and lyophilizationwhich give rise to a powder with the active ingredient plus any desiredadditional ingredient from a previously filtered sterile solutionthereof.

The pharmaceutical compositions of the invention can be suitablyadministered by means of pulse infusion, for example, with decreasingdoses of the composition. Preferably, the dose is administered by meansof injections, more preferably intravenous or subcutaneous injections,partly depending if the administration is acute or chronic.

In one embodiment, the first or second pharmaceutical compositions ofthe invention are prepared with carriers which will protect saidpolypeptide from a rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulatedadministration systems. Biodegradable biocompatible polymers such asethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters and polylactic acid can be used. The processes forpreparing said formulations will be clear for persons skilled in theart. The materials can also be commercially obtained in Alza Corporationand Nova Pharmaceuticals, Inc.

The sustained release compositions also include preparations of antibodycrystals suspended in suitable formulations which can maintain thecrystals in suspension. These preparations, when they are injected bysubcutaneous or intraperitoneal route may produce a sustained releaseeffect. Other compositions also include antibodies trapped in liposomes.The liposomes containing such antibodies are prepared by means of knownmethods such as Epstein et al., Proc. Natl. Acad. Sci. USA, (1985)82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980)77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949.

Despite the fact that Omomyc and the conjugates and fusion proteinscontaining Omomyc are capable of translocating across biologicalmembranes, the skilled person will understand that it may also beconvenient to formulate the conjugates or fusion proteins comprisingOmomyc in nanoparticles. The nanoparticles may contribute to preservethe integrity of the polypeptide in the biological fluids until itreaches the target organ. Moreover, in the case of compositionscomprising an antitumor agent, encapsulation of the composition maydecrease secondary effects caused by the antitumor agent. Lastly,nanoparticles can also be modified so as to include moieties which allowthe targeting of the nanoparticule to an organ of interest.

Thus, in another embodiment, the pharmaceutical compositions of theinvention comprise the conjugates, fusion proteins and compositionsaccording to the invention forming part of a nanoparticle.

Suitable nanoparticles that can be used in the context of the presentinvention include such nanoscale materials as a lipid-basednanoparticle, a superparamagnetic nanoparticle, a nanoshell, asemiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle,a silicon-based nanoparticle, a silica-based nanoparticle, a metal-basednanoparticle, a fullerene and a nanotube.

Targeted delivery can be achieved by the addition of ligands withoutcompromising the ability of nanoparticles to deliver their polypeptidepayloads. It is contemplated that this will enable delivery to specificcells, tissues and organs. The targeting specificity of the ligand-baseddelivery systems are based on the distribution of the ligand receptorson different cell types. The targeting ligand may either benon-covalently or covalently associated with a nanoparticle, and can beconjugated to the nanoparticles by a variety of methods as discussedherein.

Examples of proteins or peptides that can be used to targetnanoparticles include transferin, lactoferrin, TGF-β, nerve growthfactor, albumin, HIV Tat peptide, RGD peptide, and insulin, as well asothers.

The first and the second pharmaceutical compositions of the inventionmay be formulated with a pharmaceutically acceptable carrier. In apreferred embodiment, the carrier does not allow direct delivery of thefusion protein or of the composition to the cytoplasm of the cells, i.e.the carrier is not capable of fusing with the plasmatic membrane of thetarget cells. As used herein “carrier” is meant any substance thatserves to improve the delivery and the effectiveness of the activeprinciple within the pharmaceutical composition. Examples ofpharmaceutically acceptable carriers include one or more of water,saline, phosphate buffered saline, dextrose, glycerol, ethanol and thelike, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the fusion protein or of the compositions forming part of thepharmaceutical compositions. Examples of proper carriers are well knownin the literature (see for example Remington's Pharmaceutical Sciences,19th ed., Mack Publishing Company, Easton, Pa., 1995). Examples ofcarriers without limitation are a series of saccharide such as lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, andmaltitol; a series of starch such as corn starch, wheat starch, ricestarch, and potato starch; a series of cellulose such as cellulose,methyl cellulose, sodium carboxy methyl cellulose, and hydroxylpropylmethyl cellulose; and a series of filler such as gelatin andpolyvinyl pyrrolidone. In some cases, a disintegrants such ascross-linked polyvinyl pyrrolidone, agar, alginic acid, or sodiumalginate may be added.

The first and the second pharmaceutical compositions of the inventionare suitable for the administration into any type of mammal, preferablya human being.

The invention is detailed below by means of the following examples whichare merely illustrative and by no means limiting for the scope of theinvention.

EXAMPLES Materials and Methods Example 1 Expression of the OmomycConstruct in Rich Medium or in Minimal M9 Medium

For Omomyc production in rich medium, 1 L of 2YT medium containingampicillin with BL21-AI™ One Shot® Chemically competent Escherichia coli(Life technologies) transformed with the Omomyc-pET-3a construct wasinoculated, grown at 37° C. with agitation at 250 rpm until OD₆₀₀reached 0.8. The protein expression was induced with 10 mL of Arabinosesolution, incubating at 37° C. for 12 h. Alternatively, for Omomycproduction in M9 minimal medium, E. coli transformed with theOmomyc-pET3a construct was inoculated in 1 L of M9 minimal mediumcontaining chloramphenicol and ampicillin with BL21-CodonPlus(Strategene) and grown at 37° C. with agitation at 250 rpm until OD₆₀₀reached 0.8. Protein expression was induced with 1 mL of IPTG 1000×solution. The cell culture was incubated with agitation at 250 rpm at37° C. for 12 h.

The bacterial culture was centrifuged at 10,000 rpm, 4° C., 5 minutes ina SLA-1500 rotor using 250 mL bottles. The culture was centrifugedsequentially to combine the equivalent of 1 L of culture per bottle.

In order to verify the successful protein expression, 1 mL aliquot fromthe cultures was collected and its OD₆₀₀ was measured. 800 μL of saidaliquot was spined at 16,060×g for 1 min and the supernatant wasdiscarded. The pellet was suspended in a volume of IB Laemmli bufferequivalent to (0.1×OD₆₀₀ value) μL in order to ensure equal quantitiesof lysed cell extract in each sample and facilitate comparison betweencells before and after protein expression. Then the samples were mixedby vortex, sonicated for 5 seconds at maximum power, frozen in liquidnitrogen and finally, boiled for 2 minutes (repeated 3 times) prior toloading on a denaturing 16.5% acrylamide SDS-PAGE gel. Electrophoresiswas followed by Coomassie blue staining or Western blot. The IB Laemmlibuffer was optimized to allow solubilization of the inclusion bodies andto ensure adequate migration and protein separation to visualize theexpression of Omomyc.

Example 2

Purification of the Omomyc b-HLH-LZ

Cell pellets were suspended in 3 mL of Lysis buffer per gram of pelletby vortexing. 150 μL of Triton solution per gram of culture pellet wasadded. The viscosity of the suspension was reduced by sonicating on ice,at power 15 for 6×15 seconds using an ultrasonic homogenizer.

Then, the equivalent of 100 μL/g of culture pellet of bovine pancreaticDNase I was added followed by an incubation of 60 minutes at 37° C. withagitation at 50 rpm. Samples were centrifuged 20 min at 12,000×g (13,000rpm in a SS34 rotor in a Sorvall RC 5B Plus, use 35 mL centrifugebottles), 4° C. to pellet the inclusion bodies as well as high molecularweight complexes such as cell walls, ribosomes and non-degraded genomicDNA. Lipids, soluble proteins, amino acids, sugars and nucleic acidsconstitute the supernatant that was discarded after centrifugation.

The pelleted inclusion bodies ere solubilized with 15 mL of Bull crackerbuffer part A by vortexing. This acidic, high ionic strength anddenaturant buffer enables complete solubilization of the Omomycconstruct from the inclusion bodies and allows elimination of highmolecular weight complexes and residual DNA by centrifugation.

The inclusion bodies solution 1:1 was diluted with Bull cracker part B.After that, samples were centrifuged 30 min at 30,000×g (19,000 rpm in aSS34 rotor in a Sorvall RC 5B Plus) at 4° C.

The supernatant was purified by cation exchange chromatography on 5,cationic exchange columns of 5 mL (HiTrap™ SP Sepharose HP columns fromGE Healthcare or the equivalent) mounted in series on a FPLC system.First, column was washed with 2 column volumes (CV) of FPLC buffer B ata flow rate of 5 mL/min. Then, the column was equilibrated with 5 CV(125 mL) of FPLC buffer A at a flow rate of 2.5 mL/min. The supernatant(equivalent to a maximum of 9 g of culture pellet per load) was loadedat 2.5 mL/min. Immediately after loading the supernatant, column waswashed with 75 mL (3 CV) of U8 buffer, at a flow rate of 2.5 mL/min.Wash with 1 CV of buffer A. Then, 3 CV of 10% Buffer B was added to thecolumn. Omomyc elution was done with a gradient of Buffer B of 10-35% in50 mL (0.5%/mL) at 2.5 mL/min in 2.5 mL fractions. In these conditions,Omomyc eluted at ˜1.5 M NaCl (i.e. ˜30% v/v of the gradient) with apurity of 90%.

The fractions containing the pure construct were pooled and desalted on5 consecutive HiTrap™ Desalting columns from GE Healthcare (or theequivalent) equilibrated with Desalting TFA buffer, at a 3.0 mL/min flowrate. The pooled fractions were injected (maximum volume of 7.5 mL perinjection) and the elution was followed with OD₂₈₀ detector. The proteineluted within the first 15 mL after injection. The following fractionsshowing low OD₂₈₀ values and high conductivity were discarded. Thismethod allowed ˜95% recovery of 98-99% desalted protein.

The purified protein can concentrate either by centrifugation inAmicon®Ultra centrifugal filters Ultracel®-3K (Millipore™) or theequivalent, or by lyophilization. For concentration using theAmicon®Ultra, follow manufacturer's indication. Alternatively, forlyophilization, add 50% v/v acetonitrile to the desalted fractions,freeze in liquid nitrogen and lyophilize. The foamy lyophilized proteinobtained can be weighted and resolubilized to 100 μl/mg in 50% v/vacetonitrile containing 0.05% v/v TFA, aliquoted 1 to 10 mg pereppendorf and lyophilized again.

Yields around 25 mg/L of culture in rich medium and 15 mg/L of culturein minimal M9 medium.

Example 3

Omomyc Transduces into A549 Cells

The authors of the invention aimed to demonstrate that cells treatedwith Omomyc show that, even after such a short time, Omomyc alreadyreaches the nucleus.

Expression and purification of Omomyc was done as it is shown inExamples 1 and 2.

A549 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal bovine serum at 37° C. in ahumidified atmosphere containing 5% CO2. For fluorescence microscopy,20,000 A549 cells were seeded on 0.5 inch glass cover slips and grownfor 16 hours. Fresh media supplemented with Omomyc peptide (35 μM) wasadded and incubated for 2 hours at 37° C. Cells were washed 3 times withPBS and fixed in 3% paraformaldehyde, and observed under the microscope.

FIG. 1 shows that Omomyc can work as a protein transduction domaintransducing across the cellular membrane and translocating to thenucleus.

Example 4 Omomyc Reduces the Number of Viable A549 Cells and InducesApoptosis

A549 cells were incubated in 24-well plates over a period of 72 hourswith 10 μM Max* or Omomyc. Max* was obtained as described in Montagne M.et al. (Montagne M. et al. 2012. PLoS One, 7(2):e32172). Cells wereharvested and stained with Annexin V and PI according to themanufacturer protocol (Tali® Apoptosis Kit A10788, Life technologies)and the fluorescence quantified using Tali® image-based cytometer.

In this study the authors of the present invention show that Omomyc ismore efficient at reducing the number of viable A549 cells than thebHLHZ domain of Max (FIGS. 2 A and C). Consistently, the percentage ofdead/apoptotic cells is higher upon treatment with the Omomyc peptidethan with the Max* peptide (FIGS. 2B and D).

Example 5

Omomyc is More Folded than c-Myc* and More Thermally Stable than BothMax* and c-Myc* in Solution.

Thermally stable polypeptides are advantageous in those circumstanceswhen the polypeptide has to be formulated as a pharmaceuticalcomposition. Hence, the comparative thermal stability of Omomyc and Max*were determined.

The Omomyc gene was expressed as described in Example 1 and purified asin Example 2 above. The purification of Max* was carried out asdescribed in detail in Beaulieu M. E. et al., 2013 (Methods Mol Biol,1012:7-20). Briefly, BL21-DE3-pLys bacteria were transformed with apET-3a expression vector containing the Max* insert, and cultured asdescribed above. Induction was carried out with IPTG (0.6 mM) for 4hours, after which cells were harvested. After lysis with the Lysisbuffer and centrifugation at 30000×g, the solubilized protein extractwas purified using cation-exchange chromatography and desalted asdescribed above. The purified and lyophilized proteins were resuspendedin 50 mM KH₂PO₄, 50 mM KCl, 5 mM DTT (pH was adjusted using 1N KOH or 1NHCl).

Circular dichroism measurements were performed as described in BeaulieuM. E. et al., 2012 (J Mol Recognit, 25(7):414-26), using a proteinconcentration of 32 μM. The CD spectra were recorded at 20° C. and thethermal denaturation was performed at 1° C./min (FIGS. 3A and 3B),monitoring at a wavelength of 222 nm to measure the helical content(reflecting the folded, structured state) of the protein.

Example 6

Omomyc Penetrates Nuclei of Cells More Efficiently than Max*

Briefly, the purified Omomyc and Max* peptides were labelled withfluorescein-maleimide (FITC) through an engineered C-terminal cysteineresidue. After purification and assessment of the presence of a singlefluorescent label per protein molecule by mass spectrometry, differentconcentrations of protein (5 μM, 10 μM or 25 μM) were added to A549cells (K-Ras mutant lung adenocarcinoma) cultured in RPMI containing0.5% serum and incubated for 2 h at 37° C. before being washed thricewith PBS and fixed in 4% PFA for 10 min, stained with 1.5 μg/mL Hoechstfor 10 min and mounted on microscopy slides. The confocal microscopyimages showed that Omomyc penetrates the nuclei of A549 cells moreefficiently than Max* at concentrations of 5, 10 and 25 μM (data notshown). Nuclear fluorescence intensity was quantified using ImageJ, with30 to 80 cells counted per image. The quantification of the cellpenetration observed for Omomyc and Max* in A549 fixed cells is shown inFIG. 4.

The superior cell penetrating ability of Omomyc over Max* was confirmedat a concentration of 20 μM in live cells (to avoid potential artifactsof fixation) after 20 minutes of incubation. Briefly, the FITC-labelledOmomyc and Max* were added to A549 culture cells in RPMI containing 0.5%serum and incubated for 20 minutes at 37° C. before being washed thricein PBS and mounted with Hoechst-containing mounting media. The confocalmicroscopy images showed that Omomyc penetrates the nuclei of A549 livecells more efficiently than Max* at 20 μM (data not shown). Nuclearfluorescence intensity was quantified using ImageJ, with 30 to 80 cellscounted per image. The quantification of the cell penetration observedfor Omomyc and Max* in A549 live cells is shown in FIG. 5.

Example 7 Omomyc Entry Occurs Through an ATP-Dependent Process

Max* was shown to penetrate cells through an ATP-dependent process,which can be blocked lowering the temperature at 4° C. A similarmechanism is involved in Omomyc's cell penetrating ability. A549 cellswere incubated for 2 hours with 20 μM FITC-labelled peptide at 4° C. andfixed with 4% PFA, before being washed thrice with PBS and mounted onmicroscopy slides using Hoechst-containing mounting media. Confocalmicroscopy images showed that Omomyc entry can be blocked lowering thetemperature at 4° C.

Example 8

Omomyc Reduces the Total Number of Cells More Efficiently than Max*

Briefly, A549 and H1650 lung adenocarcinoma cells were incubated with 25μM Omomyc or Max* peptides in RPMI containing 5% serum and fixed at theindicated time (2 days or 4 days) with 4% PFA followed by crystal violetstaining (0.5% crystal violet in 25% methanol for 10 minutes) andwashing at least three times with water. Both Omomyc and Max* preventgrowth of cells, but Omomyc is more efficient in doing so (FIG. 6). Thecrystal violet stained cells were dissolved in 10% acetic acid and theresulting solution was diluted 1:4 in H2Odd and quantified by measuringabsorbance at OD₅₉₅. Quantification of the inhibition of proliferationby crystal violet showed that Omomyc is clearly more efficient than Max*to reduce proliferation of H1650 cells (EGFR-mutant lung adenocarcinomacells) when used at 25 μM concentration over 6 days (FIG. 7).

In order to calculate the IC₅₀ of Omomyc and Max, A549 cells weretreated with the indicated peptide concentration in culture mediacontaining 5% serum and stained with crystal violet as described above.Quantification was performed as described above. FIG. 8 shows doseresponse of A549 cells to Omomyc versus Max* showing that Omomyc has alower IC₅₀ than Max* (i.e. Omomyc needs a lower dose compared to Max* inorder to reduce the number of cells to a half). Specifically, IC₅₀ forOmomyc is 1.2 μM and IC₅₀ for Max* is 2.6 μM.

The effect of Omomyc on proliferation of cells was assayed in U87 gliomacells. U87 glioma cells were incubated with Omomyc or Max* peptides (25μM final concentration) in DMEM containing 5% serum for 48 h. Cells weretrypsinized and counted using Tali cell counter (Life technologiesInc.). FIG. 9 shows that Omomyc reduces the proliferation of U87 gliomacells more efficiently than Max*.

Example 9

Omomyc Efficiently Reaches Lung and Brain Tissue after IntranasalAdministration

Animals were treated with a single dose of 37.5 mg/kg of Omomyc or leftuntreated (treated with PBS) by intranasal administration. 10 minutesafter intranasal administration the fluorescent peptide was detected.The lung of the treated animals appears fluorescent as a consequence ofOmomyc localization (FIG. 10A). This demonstrates that the Omomycpeptide can be administered to animals by intranasal instillation and itefficiently reaches the lung. The brain of the same animals described inthe previous figure also appears fluorescent (FIG. 10B). It is apparentthat the fluorescent Omomyc peptide can pass the blood-brain barrier(BBB) and reach the brain.

Example 10 Omomyc Peptide has a Therapeutic Effect In Vivo

Administered as a peptide intranasally, Omomyc reduces proliferation ina KRas-driven lung adenocarcinoma mouse model. The inventors made use ofa mouse model of KRas driven tumorigenesis (the LSLKRasG12D model).Briefly, 8 weeks old mice were instillated with an AdenoCre virus toinduce expression of the KRas-G12D mutant protein specifically in thelungs. When the mice developed lung adenocarcinoma (18 weeks afterinfection), the animals were treated intranasally with the Omomycpeptide (15 mg/kg in 35 μL volume) daily for 3 days. Omomyc reduced theproliferative rate of tumors (Ki67 staining; FIG. 11A) and reducedcellular density (FIG. 11B).

The Omomyc peptide also reduces tumor burden in vivo after 1 weektreatment. Using the same mouse model and experimental conditions asdescribed previously, the animals were treated daily for 1 week with 15mg/kg of peptide. The % tumor area was measured using ImageJ. FIG. 12shows that Omomyc reduces tumor burden in a KRas-driven lungadenocarcinoma mouse model.

1-21. (canceled)
 22. A conjugate comprising: (i) a polypeptide of SEQ IDNO: 1, or a functionally equivalent variant thereof having a degree ofidentity with respect to SEQ ID NO: 1 greater than 60%, and (ii) achemical moiety that facilitates cellular uptake of the polypeptide orof the functionally equivalent variant thereof.
 23. The conjugateaccording to claim 22, wherein the chemical moiety that facilitatescellular uptake of the polypeptide or of the functionally equivalentvariant thereof is a cell penetrating peptide and wherein said cellpenetrating peptide and said polypeptide or the functionally equivalentvariant thereof form a fusion protein.
 24. The conjugate according toclaim 23, wherein the cell-penetrating peptide sequence is selected fromthe group consisting of GRKKRRQRRR (SEQ ID NO: 28) and RRRRRRLR (SEQ IDNO: 29).
 25. The conjugate according to claim 22, further comprising anuclear-localization signal.
 26. The conjugate according to claim 25,wherein the nuclear localization signal is selected from the groupconsisting of PKKKRKV (SEQ ID NO: 39), PAAKRVKLD (SEQ ID NO: 50) andKRPAATKKAGQAKKKK (SEQ ID NO: 40).
 27. The conjugate according to claim22, wherein the functionally equivalent variant of the polypeptide ofSEQ ID NO: 1 has a degree of identity with respect to SEQ ID NO: 1greater than 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%.
 28. A method for treating cancer comprising administering to asubject in need thereof a therapeutically effective amount of (i) apolypeptide of SEQ ID NO: 1 or a functionally equivalent variant thereofhaving a degree of identity with respect to SEQ ID NO: 1 greater than60%; or (ii) a conjugate according to claim 22, wherein the method doesnot involve the administration of the nucleic acid encoding Omomyc orthe functionally equivalent variant thereof.
 29. The method according toclaim 28, wherein the cancer is lung cancer, breast cancer or braincancer.
 30. The method according to claim 28, wherein the cancer isMyc-induced cancer.
 31. The method according to claim 28, wherein thecancer is a cancer associated with a mutation in the KRAS gene.
 32. Themethod according to claim 31, wherein the mutation in the KRAS gene is amutation at the glycine at position 12, at the glycine at position 13,or at the glutamine at position
 61. 33. The method according to claim31, wherein the mutation in the KRAS gene is selected from the groupconsisting of the G12S mutation, the G12V mutation, the G13D mutation,the G12C mutation, the G12R mutation, the G12F mutation, the G12Imutation, the G13C mutation, the G13R mutation, or the Q61L mutation.34. The method according to claim 28, wherein the functionallyequivalent variant of the polypeptide of SEQ ID NO: 1 has a degree ofidentity with respect to SEQ ID NO: 1 greater than 70%, 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
 35. The method according toclaim 28, wherein the functionally equivalent variant of the polypeptideof SEQ ID NO: 1 is a polypeptide which results from the deletion,insertion or addition of one or more amino acids with respect to thepolypeptide of SEQ ID NO:1, and/or from a conservative substitution ofone or more amino acids with respect to the polypeptide of SEQ ID NO: 1.36. The method according to claim 28, wherein the polypeptide isadministered via endovenous route or intranasal route.
 37. A compositioncomprising, together or separately: (i) a polypeptide of SEQ ID NO: 1, afunctionally equivalent variant thereof having a degree of identity withrespect to SEQ ID NO: 1 greater than 60%, or a conjugate according toclaim 22, and (ii) an antitumoral agent.
 38. The composition accordingto claim 37, wherein the functionally equivalent variant of thepolypeptide of SEQ ID NO: 1 has a degree of identity with respect to SEQID NO: 1 greater than 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99%.
 39. The composition according to claim 37, wherein theantitumoral agent is paclitaxel.
 40. A pharmaceutical compositioncomprising a pharmaceutically active amount of the conjugate accordingto claim 22 and a pharmaceutically acceptable excipient.
 41. Apharmaceutical composition comprising a pharmaceutically active amountof the composition according to claim 37 and a pharmaceuticallyacceptable excipient.
 42. A method for treatment of cancer thatcomprises administering to a subject in need thereof a therapeuticallyeffective amount of the pharmaceutical composition according to claim41.